The growth and function of anchorage dependent cells is closely tied to local microenvironmental cues surrounding the cell including the nature of the underlying substrate, the degree of cell-cell contact (both homotypic and heterotypic), paracrine signaling, and physical forces. Mooney, D.; Hansen, L.; Vacanti, J.; Langer, R; Farmer, S.; Ingber, D. J Cell Physiol 1992, 151, 497-505; Reid, L. M.; Fiorino, A. S.; Sigal, S. H.; Brill, S.; Holst, P. A. Hepatology 1992, 15, 1198-1203; Nelson, C. M.; Chen, C. S. Febs Letters 2002, 514, 238-242; Bhatia, S, N.; Balis, U. J.; Yarmush, M. L.; Toner, M. Faseb J 1999, 13, 1883-1900; Tan, J. L.; Tien, J.; Pirone, D. M.; Gray, D. S.; Bhadriraju, K.; Chen, C. S. Proceedings of the National Academy of Sciences of the United States of America. 2003, 100, 1484-1489; Galbraith, C. G.; Sheetz, M. P. Current Opinion in Cell Biology 1998, 10, 566-571. Cell adhesion to culture materials can be modulated through adsorption of extra cellular matrix (ECM) components which interact with the cell through various integrin signaling pathways. Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Science 1997, 276, 1425-1428; Flaim, C. J.; Chien, S.; Bhatia S. N. Nature Methods 2005, 2, 119-125. In addition, the degree of cell-cell interaction can influence their fate and function through both contact-mediated and soluble signals from neighboring cells. Schwartz, M. A.; Ginsberg, M. H. Nature Cell Biology 2002, 4, E65-E68. Traditional tools to address this microenvironmental parameter space are limited to bulk manipulations of the culture conditions. Kan, P.; Miyoshi, H.; Yanagi, K; Ohshima, N. Asaio J 1998, 44, M441-444. Adsorbing biomolecules such as ECM components to the substrate can modulate cell-matrix interactions. Cell-cell interactions are probed through seeding densities (higher densities increase homotypic interactions and lower densities reduce homotypic interactions), or co-cultivation with other cell types at various ratios to alter the homotypic and heterotypic interface. Hamaguchi, K.; Utsunomiya, N.; Takaki, R.; Yoshimatsu, H.; Sakata, T. Experimental Biology and Medicine 2003, 228, 1227-1233. While these techniques have yielded valuable experimental data and insight, engineering microenvironmental cues through micropatterning in high throughput biological formats enables precise experimentation not currently available using traditional techniques. Montesano, R; Mouron, P.; Amherdt, M.; Orci, L. Journal of Cell Biology 1983, 97, 935-939.
Recently, various methods to control cell-matrix and cell-cell interactions through protein and cellular micropatterning have been demonstrated. Khademhosseini, A.; Langer, R.; Borenstein, J.; Vacanti, J. P. PNAS 2006, 103, 2480-2487. Some examples include microcontact printing, microfluidic patterning, photolithographic patterning, stencil patterning, and ink-jet printing. Singhvi, R.; Kumar, A.; Lopez, G. P.; Stephanopoulos, G. N.; Wang, D. I. C.; Whitesides, G. M.; Ingber, D. E. Science 1994, 264, 696-698; Chiu D. T.; Jeon, N. L.; Huang, S.; Kane, R. S.; Wargo, C. J.; Choi, I. S.; Ingber, D. E.; Whitesides, G. M. Proceedings of the National Academy of Science 2000, 97, 2408-2413; Bhatia, S. N.; Yarmush, M. L.; Toner, M. J Biomed Mater Res 1997, 34, 189-199; Folch, A; Jo, B. H.; Hurtado, O.; Beebe, D. J.; Toner, M. Journal of Biomedical Materials Research 2000, 52, 346-353; Pardo, L.; Wilson, W. C.; Boland, T. Langmuir 2003, 19, 1462-1466. However, these techniques often require specific substrates (gold for microcontact printing or ink jet printing), are limited to simple geometries (microfluidic and stencil patterning) and flat surfaces (glass or silicon for photolithography), and cannot be utilized in high-throughput platforms such as multi-well plates.
Precise engineering of cellular microenvironments is an exciting new addition to the biologist's toolkit; however, the fabrication complexity of many techniques impedes their implementation in standard biological labs. Accordingly, a need exists for a method for etching of physisorbed biomolecules utilizing inexpensive, easily accessible off-the-shelf cell culture materials including multi-well plates, flasks, and bottles, which method would empower biological investigations through streamlined micropatterning.